The effect of growth environment on the evolution of antibiotic resistance

Sub-inhibitory levels of antibiotics impose selection pressure in favour of antibiotic resistance in bacterial species. However, it is unclear whether varying levels of sub-inhibitory concentrations have an effect on natural selection for resistance mutations. Different strengths of selection pressure could lead to a spectrum of resistance mutants arising, with different effects on bacterial fitness (benefits and costs of the mutations). These properties will affect how bacterial populations evolve during exposure to antibiotics, and when the antibiotic is removed. The effect of the growth media on the resistance evolutionary rate and fitness costs and benefits is also not well established. These could also be very different in different natural environments. In vitro tests are regularly performed in standard laboratory media, but it is unclear whether resistance evolutionary dynamics would vary in a more clinically realistic model. We will grow three species of pathogenic bacteria in the presence of clinically relevant antibiotics in different growth media: Mueller Hinton broth, artificial sputum and artificial wound fluid, and in 3D lab models of soft-tissue wound and cystic fibrosis lung biofilm. We will test whether the choice of environment has an effect on the minimum inhibitory concentrations (MICs) of the bacterial species. We will also use RNAseq and metabolomics to assess how different bacterial gene expression and metabolism is in different environments, as this could explain differences in antibiotic tolerance and differences in targets for selection for resistance. We will then evolve the bacteria in the various environments with differing sub-inhibitory concentrations of antibiotic. We will assess the frequency of de novo resistant mutants and their fitness in "home" and "away" media. Whole genome sequencing will be used to assess whether there are systematic differences in mutations that arise in the different bacterial growth conditions.